Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF
Michele Fuortes, … , Gholson J. Lyon, Carl Nathan
Michele Fuortes, … , Gholson J. Lyon, Carl Nathan
Published August 1, 1999
Citation Information: J Clin Invest. 1999;104(3):327-335. https://doi.org/10.1172/JCI6018.
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Article

Role of the tyrosine kinase pyk2 in the integrin-dependent activation of human neutrophils by TNF

Abstract

Secretion of inflammatory products from neutrophils can be induced by a combination of signals from ligated integrins and receptors for soluble, physiological agonists such as TNF. Here we identify pyk2 in primary human neutrophils; localize it to focal adhesions and podosomes; and demonstrate its tyrosine phosphorylation, activation, and association with paxillin during stimulation of adherent cells by TNF. Tyrphostin A9 emerged as the most potent and selective of 51 tyrosine kinase inhibitors tested against the TNF-induced respiratory burst. Tyrphostin A9 inhibited TNF-induced tyrosine phosphorylation of pyk2 without blocking the cells’ bactericidal activity. Wortmannin, an inhibitor of phosphatidylinositol-3-kinase, potently blocked the TNF-induced respiratory burst and selectively inhibited tyrosine phosphorylation of pyk2. Thus, pyk2 appears to play an essential role in the ability of neutrophils to integrate signals from β2 integrins and TNF receptors.

Authors

Michele Fuortes, Maxine Melchior, Hyunsil Han, Gholson J. Lyon, Carl Nathan

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Tyrphostin A9 inhibits the TNF-induced respiratory burst of human PMNs a...
Tyrphostin A9 inhibits the TNF-induced respiratory burst of human PMNs and TNF-induced tyrosine phosphorylation of pyk2 but not their bactericidal activity. (a) Inhibition of TNF-stimulated H2O2 release. PMNs were plated in FBS-coated 96-well plates and preincubated for 30 minutes with the indicated concentrations of tyrphostin A9. Cells were then unstimulated (–) or stimulated (+) either with TNF (100 ng/mL; gray bars) or PMA (100 ng/mL; filled bars), and H2O2 release was recorded 60 minutes later as mean ± SEM of triplicates. (b) Lack of inhibition of bacterial killing. PMNs were preincubated (30 minutes at 4°C) with 0 (filled squares) or 5 μM (open squares) tyrphostin A9 and then infected with 10% autologous serum-opsonized Salmonella typhimurium. Cells were lysed at indicated times and CFUs were determined. Survival of bacteria without PMNs is shown with 0 (filled circles) or 5 μM (open circles) tyrphostin A9. Mean ± SEM of triplicates; most error bars fall within the symbols. (c) PMNs were plated on FBS-coated plates, preincubated for 30 minutes in the presence or absence of tyrphostin A9 (2 μM), and then stimulated with TNF (250 ng/mL) or left untreated for 60 minutes, as indicated. Cell lysates were immunoprecipitated (IP) with anti-pyk2 antibody. Total cell lysate and immunoprecipitates were separated by reducing SDS-PAGE, transferred to nitrocellulose, and Western blotted (WB) with anti-phosphotyrosine mAb (top) or anti-pyk2 antibody (bottom) followed by ECL detection. Molecular mass markers are indicated in kilodaltons.